Phleomycin D1

Bleomycin (BLM) is used clinically in combination with a number of other agents for the treatment of several types of tumours. Members of the BLM family of drugs include zorbamycin (ZBM), phleomycin D1, BLM A2 and BLM B2. By manipulating the BLM biosynthetic machinery, we have produced two new BLM analogues, BLM Z and 6'-deoxy-BLM Z, with the latter exhibiting significantly improved DNA cleavage activity. Here we determined the DNA sequence specificity of BLM Z, 6'-deoxy-BLM Z and ZBM, in comparison with BLM, with high precision using purified plasmid DNA and our recently developed technique. It was found that ZBM had a different DNA sequence specificity compared with BLM and the BLM analogues. While BLM and the BLM analogues showed a similar DNA sequence specificity, with TGTA sequences as the main site of cleavage, ZBM exhibited a distinct DNA sequence specificity, with both TGTA and TGTG as the predominant cleavage sites. These differences in DNA sequence specificity are discussed in relation to the structures of ZBM, BLM and the BLM analogues. Our findings support the strategy of manipulating the BLM biosynthetic machinery for the production of novel BLM analogues, difficult to prepare by total synthesis; some of which could have beneficial cancer chemotherapeutic properties.

Most genetic transformation protocols for the model diatom Phaeodactylum tricornutum rely on one of two available antibiotics as selection markers: Zeocin (a formulation of phleomycin D1) or nourseothricin. This limits the number of possible consecutive genetic transformations that can be performed. In order to expand the biotechnological possibilities for P. tricornutum, we searched for additional antibiotics and corresponding resistance genes that might be suitable for use with this diatom. Among the three different antibiotics tested in this study, blasticidin-S and tunicamycin turned out to be lethal to wild-type cells at low concentrations, while voriconazole had no detectable effect on P. tricornutum. Testing the respective resistance genes, we found that the blasticidin-S deaminase gene (bsr) effectively conferred resistance against blasticidin-S to P. tricornutum. Furthermore, we could show that expression of bsr did not lead to cross-resistances against Zeocin or nourseothricin, and that genetically transformed cell lines with resistance against Zeocin or nourseothricin were not resistant against blasticidin-S. In a proof of concept, we also successfully generated double resistant (against blasticidin-S and nourseothricin) P. tricornutum cell lines by co-delivering the bsr vector with a vector conferring nourseothricin resistance to wild-type cells.

The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation. We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1. We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts. Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE.